Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder causing cognitive, memory and behavioural impairments. It is the most common cause of dementia in the elderly population affecting roughly 5% of the population above 65 years and 20% above 80 years of age. AD is characterized by an insidious onset and progressive deterioration in multiple cognitive functions. The neuropathology involves both extracellular and intracellular argyrophillic proteineous deposits. The extracellular deposits, referred to as neuritic plaques, mainly consist in amyloid beta protein (Aβ) surrounded by dystrophic neurites (swollen, distorted neuronal processes). Aβ within these extracellular deposits are fibrillar in their character with a β-pleated sheet structure. Aβ in these deposits can be stained with certain dyes e.g. Congo Red and display a fibrillar ultrastructure. These characteristics, adopted by Aβ in its fibrillar structure of neuritic plaques, are the definition of the generic term amyloid. The classic intracellular AD pathologic lesion is the neurofibrillary tangle (NFT) which consists of filamentous structures called paired helical filaments (PHFs) composed of twisted strands of hyperphosphorylated microtubule-associated protein tau. Frequent neuritic plaques and neurofibrillary tangle deposits in the brain are diagnostic criteria for AD, as carried out post mortem. AD brains also display macroscopic brain atrophy, nerve cell loss, local inflammation (microgliosis and astrocytosis) and often congophilic amyloid angiopathy (CAA) in cerebral vessel walls.
Two forms of Aβ peptides, Aβ40 and Aβ42, are the dominant species in AD neuritic plaques (Masters 1985), while Aβ40 is the prominent species in cerebrovascular amyloid associated with AD (Glenner 1984). Enzymatic activities allow Aβ to be continuously formed from a larger protein called the amyloid precursor protein (APP) in both healthy and AD afflicted subjects in all cells of the body. Two major APP processing events through β- and γ-secretase activities enables Aβ production, while a third enzyme called α-secretase activities prevents Aβ generation by cleavage inside the Aβ sequence (Selkoe, 1994; U.S. Pat. No. 5,604,102). The Aβ42 is a forty two amino acid long peptide i.e. two amino acids longer at the C-terminus, as compared to Aβ40. Aβ42 is more hydrophobic, and does more easily aggregate into larger structures of Aβ peptides such as Aβ dimers, Aβ tetramers, Aβ oligomers, Aβ protofibrils or Aβ fibrils. Aβ fibrils are hydrophobic and insoluble, while the other structures are all less hydrophobic and soluble. All these higher molecular structures of Aβ peptides are individually defined based on their biophysical and structural appearance e.g. in electron microscopy, and their biochemical characteristics e.g. by analysis with size-exclusion chromatography/western blot. These Aβ peptides, particularly Aβ42, will gradually assemble into a various higher molecular structures of Aβ during the life span. AD, which is a strongly age-dependent disorder, will occur earlier in life if this assembly process occurs more rapidly. This is the core of the “amyloid cascade hypothesis” of AD which claims that APP processing, the Aβ42 levels and their assembly into higher molecular structures is a central cause of AD. All other neuropathology of AD brain and the symptoms of AD such as dementia are somehow caused by Aβ or assembled forms thereof.
Aβ can exist in different lengths i.e. 1-39, 1-40, 1-42 and 1-43 and fragments sizes i.e. 1-28, 3-40/42, 11-40/42, 17-40/42 and 25-35. All these peptides can aggregate and form soluble intermediates and insoluble fibrils, each molecular form having a unique structural conformation and biophysical property. Monomeric Aβ1-42 for example, is a 42 amino acid long soluble and non-toxic peptide, that is suggested to be involved in normal synapse functions. Under certain conditions, the Aβ1-42 can aggregate into dimers, trimers, tetramers, pentamers up to 12-mer and higher oligomeric forms, all with its distinct physicochemical property such as molecular size, EM structure and AFM (atomic force microscopy) molecular shape. An example of a higher molecular weight soluble oligomeric Aβ form is the protofibril (Hartley 1999, Walsh 1999), which has an apparent molecular weight >100 kDa and a curvelinear structure of 4-11 nm in diameter and <200 nm in length. It has recently been demonstrated that soluble oligomeric Aβ peptides such as Aβ protofibrils impair long-term potentiation (LTP) (Hartley, 1999), a measure of synaptic plasticity that is thought to reflect memory formation in the hippocampus (Walsh 2001). Furthermore, oligomeric Arctic Aβ peptides display much more profound inhibitory effect than wtAβ on LTP in the brain, likely due to their strong propensity to form Aβ protofibrils (Klyubin 2003).
There are also other soluble oligomeric forms described in the literature that are distinctly different from protofibrils. One such oligomeric form is ADDL (Amyloid Derived Diffusible Ligand) (Lambert 1998). AFM analysis of ADDL revealed predominantly small globular species of 4.7-6.2 nm along the z-axis with molecular weights of 17-42 kDa. (Stine 1996). Another form is called ASPD (Amyloidspheroids). ASPD are spherical oligomers of Aβ1-40. Toxicity studies showed that spherical ASPD>10 nm were more toxic than lower molecular forms (Hoshi 2003). The Aβ fibril as the main neurotoxic species is inconsistent with the poor correlation between neuritic plaque density and AD dementia score and also with the modest signs of neurodegeneration in current APP transgenic mice. Soluble neurotoxic Aβ-intermediate species and their appropriate subcellular site of formation and distribution could be the missing link that will better explain the amyloid hypothesis. This idea has gained support from recent discovery of the Arctic (E693) APP mutation, which causes early-onset AD (US 2002/0162129 A1; Nilsberth et al., 2001). The mutation is located inside the Aβ peptide sequence. Mutation carriers will thereby generate variants of Aβ peptides e.g. Arctic Aβ40 and Arctic Aβ42. Both Arctic Aβ40 and Arctic Aβ42 will much more easily assemble into higher molecular structures (protofibrils) that are soluble and non-fibrillar. Thus the pathogenic mechanism of the Arctic mutation suggests that the soluble higher molecular protofibrils are causing AD.
2.1 Diagnosis of Alzheimer's Disease
2.1.1 Clinical Diagnosis
The clinical diagnosis of Alzheimer's disease (AD) is difficult to make, especially in early stages of the disease. Today, the diagnosis is based on a typical medical history combined with the exclusion of other causes of dementia. Clinical centres with high specialization can have a diagnostic accuracy of 85-90% compared with the neuropathological diagnosis. In the early stages of the disease the clinical picture is vague and definite diagnostic markers have not yet been identified (McKhann 1984). The development of biochemical diagnostic markers is important for a number of reasons: to support the clinical diagnosis, to allow clinicians to give adequate information to patients and their relatives, to initiate pharmacological treatment and care-giving, and in various aspects of clinical research.
2.1.2 Amyloid β-Peptide
Pathogenic mutations in the APP and presenilin (PS) genes have been discovered in families with early-onset AD inherited as a dominant trait (Hardy 1992). The effects of some of these mutations are now fairly well understood. The Swedish AD mutation (Mullan 1992; Axelman 1994; Lannfelt 1994) has revealed one pathogenic mechanism for the development of AD. When a cDNA construct with this mutation was transfected into human cell-lines it gave rise to approximately six times higher release of soluble Aβ (Citron 1992, Cai 1993). Furthermore, fibroblasts from individuals with the Swedish mutation secreted three times more Aβ into the media compared to fibroblasts from non-carriers (Johnston 1994). Overproduction of Aβ therefore seemed to be an important factor in the disease pathogenesis in this Swedish family. Thus, it was expected that Aβ levels measured in cerebrospinal fluid (CSF) from family members would differentiate carriers from non-carriers of the mutation. However, no difference was found in levels of total Aβ between the groups (14.5±3.3 ng/ml versus 14.9±2.3 ng/ml) (Lannfelt 1995). One explanation for this result may be that Aβ is cleared from CSF by aggregating to amyloid in the brain. However, there was a strong correlation between duration of dementia and decreasing Aβ levels. These measurements were done with antibodies recognizing soluble monomeric Aβ. With protofibril specific monoclonal antibodies more accurate measurements of the toxic species will be possible.
2.1.3 Aβ in Plasma
Aβ is found in a soluble form in plasma and other tissues (Seubert 1992), and not as previously presumed, only in the brains of AD cases. Aβ plasma levels in members of the Swedish mutation family revealed that both Aβ40 and Aβ42 were 2-3 times increased in mutation-carriers (Scheuner 1996). The proportion of Aβ42 of total Aβ was approximately 10% in both groups, which is in agreement with experiments performed in cell cultures with the Swedish mutation. Mutation-carriers below the age of expected onset of the disease had the same levels of Aβ as already affected cases. This indicates that APP mismetabolism may play an important role early in the pathogenesis of the disease.
2.1.4 Aβ42 in CSF in Alzheimer's Disease
ELISAs specifically measuring Aβ40 and Aβ42 in CSF in AD cases have given different results. Some researchers (Pirttilä 1994; Motter 1995) have found decreased Aβ42 in AD, while one group have found elevated levels in cases early in the disease progression. The most demented cases in one study had all very low levels of Aβ42. In conclusion, Aβ42 is most likely increased early in the disease process and levels of Aβ42 and Aβ40 decreased during progression of the disease. The development of accurate biochemical markers of early AD is important especially when efficient pharmacological treatments will be available in the future. Pharmacological therapy should most likely be initiated at an early stage of disease, before severe brain damage has occurred. A therapy making it possible to prevent the progression of the disease to its later stages would therefore be much desired.
2.2 Prevention and Treatment of Alzheimer's Disease
Antibodies that are specific towards different conformations of Aβ, such as Aβ fibrils (O'Nuallain 2002), micellar Aβ (Kayed 2003), ADDL (M93, M94) (Lambert 2001) have also been described.
Several pre-clinical studies in transgenic animal models have shown decreased plaque burden and improvements in memory function after active or passive immunization with antibodies raised against fibrils (Shenk 1999, Janus 2000, Morgan 2000, Weiner 2000, Sigurdsen 2001). Since fibrils are present in pathological deposits occurring late in the Alzheimer disease process, the Shenk antibodies may only be used to slow the progression of Alzheimer's disease when it has already reached its later stages.
Recently, a phase II clinical trial in Alzheimer patients with mild to moderate dementia was performed by ELAN Pharmaceuticals with their vaccine AN 1792, which is an aggregated preparation of human wtAβ42. The study had to be stopped due to side effects in 5% of the patients. The side effect was considered to be due to T-lymphocyte-induced meningioencephalitis (Nicholl 2003). The drug targets of the ELAN vaccine were insoluble fibrils found in plaques inside the brain and deposits on the brain blood vessel walls (Congophilic Amyloid Angiopathy, CAA), which are common features of Alzheimer's disease. Thus, an immune response towards insoluble fibrils could be responsible for the invasive inflammation in the brain blood vessel walls leading to meningioencephalitis.
WO02/203911 disclose the discovery of the Arctic mutation in a Swedish family leading to early onset of Alzheimer's disease (55.6 years). The Arctic mutation (Glu>Gly), which is located at position 22 in the beta amyloid peptide (Aβ), in combination with various experiments led to the insight that the Aβ peptide was much more prone to oligomerize and form protofibrils compared to wild type Aβ40. The discovery indicated for the first time that the protofibril is a central component in the disease process, and that AD could be treated by reducing the amount of protofibrils in the brain. This unique property of the Arctic mutation could then be used to generate protofibrils. WO02/203911 then suggested that said protofibrils could be used to immunize a mouse or other animal, in order to generate antibodies, after which any protofibril-specific monoclonal antibody could identified be screening. Said antibodies should then be specific towards an Aβ peptide of SEQ ID No 1 (page 7, third paragraph), i.e. a peptide carrying the Arctic mutation and having a protofibril conformation.
Thus, in view of the prior art techniques for preventing and treating Alzheimer's disease, there is a need for a technique that enables earlier detection of markers of Alzheimer's disease. If said markers could be prevented without causing negative side-effects, this would be a means to prevent and treat Alzheimer's disease at an early stage. Any treatment of Alzheimer's disease that would reduce the amount of protofibrils in the brain of AD patients, would be of significant therapeutic value.